Widely expressed CD9 and CD81 tetraspanin proteins are implicated in oocyte fertilization, somatic cell fusion, adhesion, signaling, proliferation, tumor progression, and infectious disease processes. Nonetheless, there are profound gaps in our knowledge of the basic mechanisms whereby these molecules function. Recent data indicates that CD9 and CD81 form direct, lateral associations with newly discovered IgSF proteins, EWI-2 and EWI-F, bringing them into functionally relevant complexes with a3B1 and a4B1 integrins. Our preliminary data also suggest that EWI proteins bind to counter-receptors on apposing cells, while CD9-EWI and CD81-EWI complexes interact intracellularly with cytoskeletal/cytoplasmic elements. CD9 and CD81 also interact laterally with themselves and other tetraspanins. We propose to first identify and characterize counter-receptors for EWI-2 and EWI-F. Second, we will determine functionally important residues within CD9, CD81 and EWI protein cytoplasmic tails, and then we will identify tail associated intracellular proteins. Third, direct primary associations at the core of CD9/CD81-EWI protein complexes will be disrupted. We will mutate both tetraspanin homodimerization sites, and tetraspanin-EWI protein interactions sites. Fourth, we will determine critical features of CD9 and CD81, particularly in the transmembraine regions, important for associations with a4B1 and a3B1 integrins, other tetraspanins, cholesterol, and gagliosides. Fifth, we will use our panel of tetraspanin and EWI protein mutants in functional assays, involving integrin-dependent adhesion strengthening, cell morphology, motility, fusion, and signaling. From these experiments, we sill learn key structural features of CD9 and CD81 that enable them to play a central coordinating role in multicomponent transmembrane complexes. Also, our studies will add an important new lateral dimension to the a4B1and a3B1 integrins, for which studies have historically focused mostly on ligand binding domains and cytoplasmic tails. Finally, the lessons learned here for CD9 and CD81 will be applicable to other tetraspanins, which also contribute to a wide variety of physiological activities.